PROJECT SUMMARY Alcohol consumption is prevalent in persons with Hepatitis C virus (HCV) infection. However, studies relating alcohol consumption with HCV treatment outcomes, like adherence and efficacy, are limited. This is partly due to the exclusion of those with heavy alcohol use from clinical trials and in practice. Studies examining the relationship between alcohol use and treatment outcomes mainly rely on self-reported alcohol measures. Self- report consistently underrepresents alcohol consumption in this population, thus an objective measure of alcohol consumption is needed. Phosphatidylethanol (PEth) is an ideal objective measure of alcohol consumption. PEth is 100% specific for alcohol consumption with an approximately two-week window of detection and dose proportionality. Through the work proposed, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify PEth in dried blood spots (DBS) will be developed and validated. This method will be used to quantify alcohol consumption in sixty alcohol and drug users with HCV infection treated with ledipasvir/sofosbuvir (LDV/SOF 400 mg/90 mg, Harvoni) in the INCLUD trial (NCT 02573376). The association between PEth concentrations in 360 DBS samples measured throughout HCV treatment and objective measures of adherence will be established. Objective measures of adherence include directly observed therapy (DOT) and wirelessly observed therapy (WOT) data, as well as cumulative sofosbuvir adherence in DBS as quantified by intracellular concentrations of this prodrug?s active form, 007-triphosphate. This application will test the hypothesis that increased alcohol consumption as defined by PEth concentrations will result in decreased antiviral adherence. In the course of method development and validation, the impact of various sample collection, storage, and unique DBS factors on PEth concentrations in DBS will be evaluated in order to ensure the most accurate quantification of this biomarker. Mixed model analyses will then be performed using longitudinal data to elucidate the relationship between alcohol use and antiviral adherence. Expansion of HCV treatment is essential to eliminate this disease. An NIAAA mission is to develop tools to generate evidence-based information about alcohol and public health in order to facilitate implementation of treatment interventions. This work will encourage HCV treatment by providing objective data on the association between alcohol use and HCV treatment adherence. PEth interpretation will be informed and improved through use of a fully validated DBS-based method. Findings will enhance the use of quantitative biomarkers for measuring alcohol consumption in the HCV field and beyond.